The long term goals of the proposed research are to define the mechanism of action and essential function of small heat shock/alpha-crystallin proteins (sHsps). The sHsps (16 to 42 kDa) are a ubiquitous class of chaperones that are defined by approximately 90 amino acid C-terminal alpha-crystallin domain and that typically assemble into native oligomers of 9 to >30 subunits, depending on the protein. Current models propose that sHsps act to prevent irreversible substrate-denaturation by binding substrates and keeping them in a conformational state suitable for refolding by ATP-dependent chaperones. Thereby they can mediate protection against many forms of stress. The mechanism of this protective interaction with substrates is poorly understood, and defined targets of the sHsps still need to be identified. Understanding sHsp function is important to stress responses, disease states, normal development and aging, all of which involve sHsp expression. Consistent with the model for their chaperone activity, sHsps have been increasingly linked to pathological conditions in which protein aggregation occurs. We have determined the first X-ray structure (2.65 Angstrom) of an eukaryotic sHsp, providing a structural context for further mechanistic studies. Robust in vitro assays for chaperone activity of sHsps have been developed in the laboratory. In addition, we have established a genetic system in the cyanobacterium Synechocystis sp. 6803, in which deletion or point mutation of the single sHsp gene causes temperature sensitivity. The ability to perform in vivo functional studies in this homologous system is unique to the field of sHsp/alpha-crystallin research and provides a new approach to studying the role of sHsps in protection of cells from stress. With these biochemical and genetic resources, the following specific aims will be addressed. Aim 1: To define the sHsp-substrate binding sites and the organization of sHsp/substrate complexes. Aim 2: To determine how sHsps affect substrate function in vivo. Aim 3: To define the substrate binding species of the sHsp. Aim 4: To extend our knowledge of sHsp mechanisms by examining the structure and activity of diverse members of the sHsp family. The proposed experiments will greatly advance our understanding of sHsp function and provide new insight into the role of sHsps in disease and normal development.